WO2023080137A1 - 標的物質の検出方法 - Google Patents

標的物質の検出方法 Download PDF

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Publication number
WO2023080137A1
WO2023080137A1 PCT/JP2022/040892 JP2022040892W WO2023080137A1 WO 2023080137 A1 WO2023080137 A1 WO 2023080137A1 JP 2022040892 W JP2022040892 W JP 2022040892W WO 2023080137 A1 WO2023080137 A1 WO 2023080137A1
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Prior art keywords
nucleic acid
stranded nucleic
substance
target substance
specific binding
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PCT/JP2022/040892
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English (en)
French (fr)
Japanese (ja)
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祐二 久保
洋一 牧野
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凸版印刷株式会社
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Application filed by 凸版印刷株式会社 filed Critical 凸版印刷株式会社
Priority to CN202280071151.9A priority Critical patent/CN118140141A/zh
Priority to JP2023558041A priority patent/JPWO2023080137A1/ja
Priority to EP22889957.1A priority patent/EP4428533A1/en
Publication of WO2023080137A1 publication Critical patent/WO2023080137A1/ja
Priority to US18/652,258 priority patent/US20240279717A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the present invention relates to a method for detecting target substances. More particularly, the present invention relates to a target substance detection method, a complex, and a target substance detection kit.
  • This application claims priority to Japanese Patent Application No. 2021-180020 filed in Japan on November 4, 2021, the content of which is incorporated herein.
  • the sample solution is divided into an extremely large number of minute solutions. Then, the signal from each minute solution is binarized, only whether or not the target substance exists is determined, and the number of molecules of the target substance is counted. Digital measurement can dramatically improve detection sensitivity and quantification compared to conventional ELISA, real-time PCR, and the like.
  • Non-Patent Document 1 describes a microwell array having channels for supplying microwells and reagents, and describes that digital ELISA was performed using the microwell array.
  • Patent Document 1 Non-Patent Documents 2 and 3 report Proximity Ligation Assay (PLA) and Proximity Extension Assay (PEA), which are methods for detecting proteins using antibodies modified with oligonucleotides. . These methods utilize the PCR method for detection.
  • PHA Proximity Ligation Assay
  • PEA Proximity Extension Assay
  • Kan C. W., et al. Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated array polymer assemblies., Lab on a Chip, 12 (5), 977-985, 2012.
  • Mohammed H., et al. Approaches for Assessing and Discovering Protein Interactions in Cancer., Mol Cancer Res, 11 (11), 1295-1302, 2013.
  • Assarsson E., et al. Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability., PLoS One, 9 (4), e95192, 2014.
  • Non-Patent Document 1 PSA to be detected is bound to magnetic beads, then the beads are washed, then the beads are reacted with a biotin-labeled antibody, and then the beads are washed. followed by reacting the beads with a solution containing streptavidin- ⁇ -galactosidase, washing the beads, then suspending the beads in a buffer containing the substrate, and then introducing the beads into the well array. It is described that it was detected by Thus, complicated pretreatment may be required to detect target substances by digital ELISA. SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a technique for detecting a target substance without performing a washing step.
  • the present invention includes the following aspects.
  • a method for detecting a target substance in a sample wherein the sample, a first specific binding substance for the target substance labeled with a first single-stranded nucleic acid fragment, and a second A second specific binding substance for the target substance labeled with a single-stranded nucleic acid fragment of is introduced, so that when the target substance is present in the sample, the target substance A complex containing the specific binding substance and the second specific binding substance is formed, and at least a portion of the first single-stranded nucleic acid fragment and at least a portion of the second single-stranded nucleic acid fragment hybridize.
  • Methodhod. The method according to [1], wherein the volume of the well is 10 fL to 100 pL.
  • the first specific binding substance recognizes the first binding site of the target substance
  • the second specific binding substance recognizes the second binding site of the target substance
  • the distance between the first binding site and the second binding site is such that at least a portion of the first single-stranded nucleic acid fragment and at least a portion of the second single-stranded nucleic acid fragment
  • the method of [3] which is a distance that allows hybridization.
  • [5] The method according to any one of [1] to [4], wherein each of the first single-stranded nucleic acid fragment and the second single-stranded nucleic acid fragment has a base length of 10 to 200 bases.
  • [6] further comprising the step of sealing the opening of the well after introducing the sample, the first specific binding substance, and the second specific binding substance into the well, [1 ] to [5].
  • the first specific binding substance that does not bind to the target substance and/or the second specific binding that does not bind to the target substance in the well whose opening is sealed The method of [6], comprising the substance. [8] of [1] to [7], wherein the target substance is one selected from the group consisting of proteins, sugar chains, nucleic acids, lipid membrane structures, bacteria, viruses and cells, or a composite substance thereof; Any method described. [9] Each of the first specific binding substance and the second specific binding substance is one selected from the group consisting of antibodies, lectins and substances that bind to lipid membranes, [1 ] to [8].
  • a target substance, a first specific binding substance for the target substance labeled with a first single-stranded nucleic acid fragment, and a second single-stranded nucleic acid fragment for the target substance labeled comprising a second specific binding substance, wherein at least a portion of the first single-stranded nucleic acid fragment and at least a portion of the second single-stranded nucleic acid fragment hybridized to form a double-stranded nucleic acid; Complex.
  • a target substance detection kit comprising a well array having a plurality of wells, a first specific binding substance for the target substance labeled with a first single-stranded nucleic acid fragment, and a second comprising a second specific binding substance for said target substance labeled with a single-stranded nucleic acid fragment of [13]
  • the kit according to [12] further comprising a sealing liquid that seals the opening of the well.
  • FIG. 1 is a schematic diagram illustrating a method for detecting a target substance.
  • FIG. 2 is a schematic cross-sectional view showing an example of a fluidic device.
  • FIG. 3 is a schematic cross-sectional view showing an example of a fluidic device.
  • FIG. 4 is a schematic cross-sectional view showing an example of a fluidic device.
  • FIG. 5 is a schematic cross-sectional view showing an example of a fluidic device.
  • FIG. 6 is a schematic cross-sectional view showing an example of a fluidic device.
  • FIG. 7 is a schematic cross-sectional view showing an example of a fluidic device.
  • FIG. 8 is a schematic diagram illustrating an example of the Invasive Cleavage Assay (ICA) method.
  • ICA Invasive Cleavage Assay
  • FIG. 9 is a graph showing the results of Experimental Example 1.
  • FIG. 10 is a graph showing the results of Experimental Example 1.
  • FIG. 11 is a graph showing the results of Experimental Example 1.
  • FIG. 12 is a microscope image showing the results of Experimental Example 2.
  • FIG. 13 is a graph showing the results of Experimental Example 2.
  • FIG. 14 is a graph showing the average value ⁇ standard deviation of the brightness of the wells when each concentration of the target substance was detected in Experimental Example 2.
  • FIG. FIG. 15 is a graph showing the number of wells exhibiting luminance equal to or higher than the set value when target substances of various concentrations were detected in Experimental Example 2.
  • FIG. 16 is a graph showing the results of immuno-ICA reaction using oligonucleotide-modified antibodies bound to DNA1 and DNA2 in Experimental Example 3.
  • FIG. 17 is a graph showing the results of immuno-ICA reaction using oligonucleotide-modified antibodies bound to DNA2 and DNA3 in Experimental Example 3.
  • FIG. 18 is a graph showing the results of immunoICA reaction using oligonucleotide-modified antibodies to which DNA2 and DNA4 were bound in Experimental Example 3.
  • FIG. 19 is a graph showing the results of immuno-ICA reaction using oligonucleotide-modified antibodies bound to DNA2 and DNA5 in Experimental Example 3.
  • FIG. 20 is a graph showing the results of immuno-ICA reaction using oligonucleotide-modified antibodies to which DNA2 and DNA5 were bound in Experimental Example 4.
  • the present invention provides a method for detecting a target substance in a sample, wherein the well contains the sample, a first single-stranded nucleic acid fragment labeled with a first specific binding to the target substance.
  • a method is provided for indicating the presence of a target substance.
  • FIG. 1 is a schematic diagram explaining the method of this embodiment.
  • a sample a first specific binding substance 120 labeled with a first single-stranded nucleic acid fragment 121, and a second single-stranded nucleic acid
  • a second specific binding substance 130, labeled with fragment 131 is introduced into the wells.
  • target substance 110 is present in the sample, complex 100 including target substance 110, first specific binding substance 120 and second specific binding substance 130 is formed.
  • double-stranded nucleic acid 140 is detected. If the formation of double-stranded nucleic acid 140 is detected, then target material 110 is said to be present.
  • the method of this embodiment may be applied to samples that do not contain the target substance 110, in which case the formation of the double-stranded nucleic acid 140, ie the presence of the target substance 110, is not detected. Detection of formation of double-stranded nucleic acid 140 will be described later.
  • first specific binding substance 120 recognizes first binding site 111 of target substance 110 .
  • Second specific binding substance 130 also recognizes second binding site 112 of target substance 110 .
  • first binding site 111 and second binding site 112 may be different binding sites.
  • the target substance is a protein
  • samples include biological samples and environmental samples.
  • Biological samples are not particularly limited, and include serum, plasma, urine, cell culture medium, and the like.
  • environmental samples include river water and industrial wastewater.
  • the target substance 110 may be one selected from the group consisting of proteins, sugar chains, nucleic acids, lipid membrane structures, bacteria, viruses and cells, or a composite substance thereof.
  • the protein may be a glycoprotein or a membrane protein.
  • a protein may be a complex substance. Complex substances include, for example, glycoproteins in which sugar chains and proteins are bound, fusion proteins in which multiple proteins are fused, and the like.
  • Lipid membrane structures include vesicles composed of artificial or natural lipids, such as liposomes, exosomes and intracellular organelles. In detecting lipid membrane structures, bacteria, viruses and cells, proteins, sugar chains, nucleic acids, etc. present in these target molecules may be detected.
  • the distance between the first binding site 111 and the second binding site 112 is at least part of the first single-stranded nucleic acid fragment 121 and at least one of the second single-stranded nucleic acid fragment 131.
  • the distance is such that the moieties can hybridize.
  • the hybridizable distance is preferably a distance close to a position where the first single-stranded nucleic acid fragment 121 and the second single-stranded nucleic acid fragment 131 can hybridize.
  • the distance between the first binding site 111 and the second binding site 112 is preferably 3-10 nm, more preferably 3.5-9 nm. is more preferable.
  • the angle between the first binding site 111 and the second binding site 112 with respect to the center of the target substance 110 is 30 to 120°.
  • the distance between the first binding site 111 and the second binding site 112 is between 4.14 and 8.66 nm.
  • the distance between the first binding site 111 and the second binding site 112 is defined as the shortest distance between the first binding site 111 and the second binding site 112 along the surface of the target substance 110. .
  • the base length of the first single-stranded nucleic acid fragment 121 may be 10 to 200 bases.
  • the base length of the second single-stranded nucleic acid fragment 131 may also be 10 to 200 bases.
  • the length of the double-stranded nucleic acid 140 formed by hybridizing at least part of the first single-stranded nucleic acid fragment 121 and at least part of the second single-stranded nucleic acid fragment 131 is 7 to 30. It may be about a base, or it may be 7 to 20 bases.
  • the hybridizing regions of the first single-stranded nucleic acid fragment 121 and the second single-stranded nucleic acid fragment 131 may include the ends of each single-stranded nucleic acid fragment.
  • the hybridizing region of the first single-stranded nucleic acid fragment 121 and the second single-stranded nucleic acid fragment 131 may not include the end of the single-stranded nucleic acid fragment. It may be located at ⁇ 13 bases. From the viewpoint of increasing detection sensitivity, the hybridizing regions of the first single-stranded nucleic acid fragment 121 and the second single-stranded nucleic acid fragment 131 preferably include the ends of the respective single-stranded nucleic acid fragments.
  • the method of this embodiment is suitable for detection of target substances by digital measurement.
  • the wells form a well array in which a plurality of wells are arranged.
  • the well array is preferably arranged within the channel of the fluidic device.
  • FIG. 2 is a schematic cross-sectional view showing an example of a fluidic device.
  • the fluidic device 200 includes a substrate 210 and a lid member 220 arranged to face the substrate 210 .
  • the lid member 220 has a convex portion 221 and the tip of the convex portion 221 is in contact with the substrate 210 .
  • well array 240 is formed integrally with substrate 210 on one surface of substrate 210 .
  • the surface of substrate 210 on which well array 240 is molded faces lid member 220 .
  • Well array 240 has a plurality of wells 241 .
  • the lid member 220 may be welded or adhered to the substrate 210 .
  • the well 241 is open on the surface of the substrate 210.
  • the shape, size, and arrangement of the wells 241 are not particularly limited, but one target substance is preferably introduced into one well 241 .
  • the well 241 is preferably a microwell with a small volume.
  • the volume of one well 241 may be on the order of 10 fL to 100 pL.
  • a plurality of wells 241 of the same shape and size form a well array 240 .
  • the same shape and same size means that they have the same shape and the same capacity to the extent required for performing digital measurement, and variations to the extent of manufacturing error are allowed.
  • the diameter of the well 241 may be, for example, approximately 1 to 10 ⁇ m.
  • the well 241 may have a depth of about 1 to 10 ⁇ m, for example.
  • the arrangement of the wells 241 is not particularly limited, and may be, for example, arranged in a triangular lattice, may be arranged in a square lattice, or may be arranged at random.
  • a space is formed between the well array 240 and the lid member 220 due to the presence of the protrusions 221 .
  • the space constitutes the channel 230 .
  • the channel 230 functions as a path for feeding a liquid in which a target substance, a first specific binding substance, a second specific binding substance, etc. are dispersed, and a sealing liquid, which will be described later.
  • the shape, structure, capacity, and the like of the channel 230 are not particularly limited, but the height of the channel 230 (that is, the surface of the substrate 210 facing the cover member 220 and not formed with the well array, The distance between the surface of the lid member 220 facing the substrate 210) may be, for example, 500 ⁇ m or less, may be, for example, 300 ⁇ m or less, may be, for example, 200 ⁇ m or less, or may be, for example, 100 ⁇ m. It may be below.
  • the convex portion 221 may be formed integrally with the lid member 220 .
  • the lid member 220 can be molded into a plate-like shape having the protrusions 221 by molding a thermoplastic resin fluid using a molding die, for example.
  • the lid member 220 may be formed with a reagent introduction port 222 and a reagent discharge port 223 .
  • the lid member 220 When the lid member 220 has the protrusions 221, the lid member 220 and the substrate 210 are overlapped so that the protrusions 221 are in contact with the surface of the substrate 210 on which the wells 241 are opened. As a result, the space between the lid member 220 and the substrate 210 becomes the channel 230 .
  • the lid member 220 and the substrate 210 may be welded by laser welding or the like.
  • FIG. 5 is a schematic cross-sectional view showing an example of a fluidic device.
  • fluidic device 500 includes substrate 210 and wall member 510 .
  • well array 240 is formed integrally with substrate 210 on one surface of substrate 210 .
  • Well array 240 has a plurality of wells 241 .
  • the fluidic device 500 mainly differs from the above-described fluidic device 200 in that it does not have a lid member 220 . Therefore, the fluidic device 500 does not have flow channels.
  • the lid member 220 and the convex portion 221 are integrally molded.
  • the lid member 220 and the projection 221 may be formed separately.
  • the well array 240 is formed integrally with the substrate 210 on one surface of the substrate 210 .
  • the well array does not have to be integrally molded with substrate 210 .
  • a well array 240 molded separately from the fluidic device may be placed on the substrate 210 of the fluidic device.
  • a resin layer may be laminated on the surface of the substrate 210 and a well array may be formed in the resin layer by etching or the like.
  • the substrate 210 is formed using resin, for example.
  • resin for example.
  • the type of resin is not particularly limited, it is preferably a resin that is resistant to reagents and sealing liquids.
  • the signal to be detected is fluorescence, it is preferable to use a resin with little autofluorescence.
  • resins include, but are not limited to, cycloolefin polymers, cycloolefin copolymers, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesins and amorphous fluororesins.
  • a plurality of wells 241 may be formed on one surface of the substrate 210 in the plate thickness direction.
  • Methods of forming wells using resin include injection molding, thermal imprinting, optical imprinting, and the like.
  • a well array may be formed by laminating a fluororesin on the substrate 210 and processing the fluororesin by etching or the like.
  • a fluorine resin for example, CYTOP (registered trademark) (Asahi Glass) or the like can be used.
  • the material of the lid member 220 is preferably a resin with low autofluorescence, and may be, for example, a thermoplastic resin such as cycloolefin polymer or cycloolefin copolymer.
  • the lid member 220 may be made of a material that does not transmit light having a wavelength in the vicinity of the wavelength detected during fluorescence observation of the signal, or may be made of a material that completely does not transmit light.
  • the lid member 220 may be made of thermoplastic resin to which carbon, metal particles, or the like is added.
  • the method of the present embodiment is a method for detecting a target substance in a sample, and a well 241 contains a first specific binding substance 120 for a target substance 110 labeled with a sample and a first single-stranded nucleic acid fragment 121.
  • a target substance can be detected without performing a washing step.
  • the reagent liquid L210 is introduced from the introduction port 222 of the fluidic device 200 and sent to the channel 230.
  • Reagent liquid L210 is a liquid in which a sample, first specific binding substance 120 and second specific binding substance 130 are dispersed, and also contains a reagent for detecting formation of double-stranded nucleic acid 140.
  • FIG. The reagent solution L210 is a reagent for detecting the formation of the first specific binding substance 120, the second specific binding substance 130, the target substance 110, and the double-stranded nucleic acid 140 when the target substance 110 is contained in the sample. including.
  • the reagent liquid L210 sent to the channel 230 contacts the well array 240.
  • a reagent solution L210 is accommodated inside the well 241 . This results in wells 241 containing first specific binding substance 120, second specific binding substance 130 and, if present, target substance 110, and reagents for detecting the formation of double-stranded nucleic acid 140. is introduced.
  • the reagent solution L210 can be prepared by the first method below.
  • the sample, the first specific binding substance 120 and the second specific binding substance 130 are mixed, and the target substance 110 that may be contained in the sample and the first specific binding substance 120 and the second specific binding substance 130 are combined.
  • Antigen-antibody reaction is performed.
  • the antigen-antibody reaction is carried out, for example, at 25-37° C. for 30-180 minutes.
  • a reagent for detecting the formation of double-stranded nucleic acid 140 is added, and ICA reaction is performed.
  • the ICA reaction is carried out, for example, at 60-70° C. for 10-60 minutes.
  • the mixed liquid after completion of the ICA reaction can be used as the reagent liquid L210.
  • the reagent solution L210 can be prepared by the second method below.
  • a sample, first specific binding substance 120 and second specific binding substance 130 are mixed to prepare an antigen-antibody reaction solution.
  • a reagent for detecting the formation of the double-stranded nucleic acid 140 is added to the antigen-antibody reaction solution to prepare a mixed solution. This mixture is maintained at, for example, 25-37° C. for 30-180 minutes and then at 60-70° C. for 10-60 minutes to complete the ICA reaction.
  • the mixed liquid after completion of the ICA reaction can be used as the reagent liquid L210.
  • the number of target substances 110 introduced into one well 241 is not particularly limited, preferably one or less, that is, zero or one target substance 110 is introduced into one well 241 .
  • detection of the target substance 110 can be performed in units of one, that is, digital measurement is possible. Also, it is not necessary to introduce the target substance 110 into all wells of the well array.
  • the means for introducing the target substance 110 into the well is not particularly limited, and an appropriate means can be selected according to the selected target substance 110.
  • an appropriate means can be selected according to the selected target substance 110.
  • the target substance 110 is sedimented within the fluidic device (which may be within the channel) by its own weight and distributed to the wells.
  • a substance that captures the target substance 110 also referred to as a captured substance
  • the liquid may be fed.
  • the step of binding the captured substance to the target substance 110 can be performed at any point in the method of the present embodiment. For example, this step may be performed by contacting the target substance 110 with the capture in the sample tube prior to the step of introducing the target substance 110 into the wells 241 .
  • the target substance 110 may be introduced into the well after the capture substance is introduced into the well 241, and the capture substance and the target substance 110 may be brought into contact within the well.
  • a captured substance is a substance that can capture the target substance 110 .
  • the capture may be, for example, a conjugate between a solid phase and a specific binding substance for target substance 110 .
  • Solid phases include particles, films and substrates.
  • the number of specific binding substances for the target substance 110 may be one, or two or more. For example, there may be three types, four types, or five or more types.
  • the particles are not particularly limited, and include polymer particles, magnetic particles, glass particles, and the like. Particles are preferably surface-treated particles to avoid non-specific adsorption. Particles having a functional group such as a carboxyl group on the surface are preferred for immobilizing a specific binding substance. More specifically, JSR's product name "Magnosphere LC300" or the like can be used.
  • cells to which the virus can adhere may be used as traps.
  • Specific binding substances in the first specific binding substance 120, the second specific binding substance 130, and the captured substance include antibodies, antibody fragments, aptamers, and the like.
  • antibody fragments include Fab, F(ab') 2 , Fab', single-chain antibodies (scFv), disulfide-stabilized antibodies (dsFv), dimerized V region fragments (diabody), peptides containing CDRs, and the like. be done.
  • Antibodies may be monoclonal antibodies or polyclonal antibodies. Alternatively, it may be a commercially available antibody.
  • the specific binding substance when the target substance 110 contains a sugar chain, the specific binding substance may be a lectin. Moreover, when the target substance 110 includes a lipid membrane, the specific binding substance may be a substance that has binding properties to the lipid membrane. Substances that bind to lipid membranes include, for example, hydrocarbons such as hexanediol and membrane proteins such as transmembrane proteins. Membrane proteins include, for example, ⁇ -hemolysin.
  • Examples of methods for labeling specific binding substances with single-stranded nucleic acid fragments include methods using cross-linking agents.
  • a single-stranded nucleic acid fragment may be labeled with a specific binding substance via a linker molecule.
  • the linker is not particularly limited, and examples thereof include polyethylene chains, hydrocarbon chains, peptides, and the like.
  • a single-stranded nucleic acid fragment may be DNA or RNA. It may also contain artificial nucleic acids such as BNA and LNA.
  • the method for immobilizing the specific binding substance on the particle surface is not particularly limited, and includes a method using physical adsorption, a method using chemical binding, a method using avidin-biotin binding, and binding of protein G or protein A to an antibody. and the like.
  • Physical adsorption methods include methods in which a specific binding substance is immobilized on the particle surface through hydrophobic interaction or electrostatic interaction.
  • Methods using chemical bonding include a method using a cross-linking agent. For example, when the surface of a particle has a hydroxyl group, the carboxyl group of the specific binding substance is reacted with a cross-linking agent to form an active ester, and then the hydroxyl group is reacted with the ester group to convert the specific binding substance into the particle. It can be immobilized on a surface. In addition, it is preferable to provide a spacer between the specific binding substance and the particle surface so as not to inhibit the ability of the specific binding substance to recognize the target molecule.
  • the target substance 110 when the target substance 110 is introduced into the well 241 using the capture substance, the combined body of the capture substance and the target substance 110 is obtained under the condition that 0 or 1 target substance 110 is captured by one capture substance. is preferably formed. Furthermore, one well 241 is preferably configured to introduce 0 or 1 trapped substance. This enables digital measurement.
  • a complex 100 containing them is formed, and at least part of the first single-stranded nucleic acid fragment 121 and At least a portion of second single-stranded nucleic acid fragment 131 hybridizes to form double-stranded nucleic acid 140 .
  • the formation of complexes 100 may occur in the sample tube before the sample, first specific binding substance 120 and second specific binding substance 130 are introduced into the wells, or may occur in wells 241. may be broken.
  • the target substance may consist of two molecules and may be detected using a nucleic acid-labeled specific binding substance specific to each molecule. In this case, it can be determined whether the two molecules are in a bound state. Further, when the two molecules form a homodimer, the nucleic acid-labeled first specific binding substance and the nucleic acid-labeled second specific binding substance have the same epitope may be recognized.
  • a step of sealing the opening of the well 241 may be performed.
  • the method of sealing the opening of the well 241 is not particularly limited as long as the liquid contained in one well 241 and the liquid contained in another well 241 do not mix with each other.
  • the well 241 may be sealed by covering the opening of the well 241 with a sealing liquid.
  • the opening of the well 241 may be sealed by laminating a plate-like member such as a glass plate.
  • the sealing liquid L220 is sent from the introduction port 222 of the lid member 220 to the channel 230 between the substrate 210 and the lid member 220.
  • the sealing liquid L220 sent to the channel 230 contacts the well array 240.
  • the sealing liquid L220 pushes away and replaces the reagent liquid L210 not contained in the well 241 among the reagent liquid L210 sent to the channel 230 .
  • the sealing liquid L220 individually seals the plurality of wells 241 containing the reagent liquid L210 containing the target substance 110, and the wells 241 become independent reaction spaces (micropartitions 242).
  • FIG. 4 shows that all of the wells 241 of the well array 240 are sealed with a sealing liquid L220 to form sealed wells (ie, microcompartments) 242.
  • FIG. 4 shows that all of the wells 241 of the well array 240 are sealed with a sealing liquid L220 to form sealed wells (ie, microcompartments) 242.
  • Lipids forming a lipid bilayer membrane include, for example, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) and Examples thereof include, but are not limited to, mixtures thereof.
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DOPG 1,2-dioleoyl-sn-glycero-3-phosphoglycerol
  • Wells 242 sealed by any of the sealing methods described above contain first specific binding substance 120 not bound to target substance 110 and/or second specific binding substance 120 not bound to target substance 110 . It may also contain a binding substance 130 .
  • the sealing liquid is a liquid capable of forming droplets (also called minute droplets) by individually sealing the liquids introduced into the plurality of wells 241 so that they do not mix with each other.
  • the sealing liquid is preferably an oily solution, more preferably an oil.
  • oil fluorine-based oil, silicone-based oil, hydrocarbon-based oil, or a mixture thereof can be used. More specifically, the product name "FC-40" manufactured by Sigma can be used.
  • FC-40 (CAS number: 86508-42-1) is a fluorinated aliphatic compound with a specific gravity of 1.85 g/mL at 25°C.
  • Detection process Subsequently, the formation of double-stranded nucleic acid 140 is detected. Detection of the formation of double-stranded nucleic acid 140 is preferably performed using a signal amplification reaction.
  • Signal amplification reactions include, for example, Invasive Cleavage Assay (ICA).
  • the ICA reaction is related to the principle that signal amplification proceeds through a cycle of two reactions: (1) complementary binding between nucleic acids and (2) recognition and cleavage of the triplex structure by an enzyme.
  • the ICA reaction is less affected by reaction cycle inhibition by contaminants. Therefore, by using the ICA reaction, the target substance 110 can be detected with high accuracy.
  • the reagent liquid L210 (the liquid containing the target substance 110, the first specific binding substance 120 and the second specific binding substance 130) contains reaction reagents necessary for the ICA reaction. .
  • Reaction reagents necessary for the ICA reaction include ICA reaction reagents such as flap probes, flap endonuclease (FEN), and fluorescent substrates.
  • a flap probe is a nucleic acid fragment designed to hybridize to the first single-stranded nucleic acid fragment 121 or the second single-stranded nucleic acid fragment 131 to form a double-stranded nucleic acid 140 and a flap structure.
  • FIG. 8 is a schematic diagram explaining an example of the ICA method.
  • a double-stranded nucleic acid 140 formed by hybridizing at least a portion of the first single-stranded nucleic acid fragment 121 and at least a portion of the second single-stranded nucleic acid fragment 131 by the ICA method. to detect
  • the flap probe is hybridized to the first single-stranded nucleic acid fragment 121 or the second single-stranded nucleic acid fragment 131.
  • flap probe 810 hybridizes to first single-stranded nucleic acid fragment 121 .
  • a first flap portion 811 is formed.
  • the first flap site 811 is reacted with FEN, the first flap site 811 is cleaved and a nucleic acid fragment 811 is generated. Nucleic acid fragment 811 then hybridizes to a fluorescent substrate (nucleic acid fragment 820 ) to form second flap region 821 .
  • a fluorescent substance F is bound to the 5' end of the nucleic acid fragment 820, and a quenching substance Q is bound several bases 3' from the 5' end of the nucleic acid fragment 820.
  • the second flap site 821 is reacted with FEN, the second flap site 821 is cleaved and a nucleic acid fragment 821 is produced.
  • the fluorophore F is separated from the quencher Q, generating a fluorescence signal. Formation of the double-stranded nucleic acid 140 can be detected by detecting this fluorescence signal.
  • the reagent liquid L210 a common liquid used in biochemical analysis performed using a fluidic device can be used, preferably an aqueous solution. Further, the reagent liquid L210 may contain a surfactant or the like to make it easier to enclose the liquid in the well.
  • the isothermal enzymatic reaction causes the fluorescent substance F to be liberated from the quenching substance Q and respond to the excitation light. to emit a predetermined fluorescence signal.
  • a known appropriate method can be selected according to the type of signal to be detected. For example, when observing a fluorescence signal, excitation light corresponding to a fluorescent substance is applied to the minute compartment 242, and fluorescence emitted by the fluorescent substance is observed. For example, as shown in FIG. 4, a given reaction is performed within microcompartment 242 and the signal generated is observed. Microcompartment 242R is the well in which signal was detected and microcompartment 242 is the well in which no signal was detected.
  • a reagent liquid L210 is introduced into the fluidic device 500 as shown in FIG.
  • Reagent solution L210 is a liquid in which target substance 110, first specific binding substance 120, and second specific binding substance 130 are dispersed, and also contains a reagent for detecting formation of double-stranded nucleic acid 140.
  • concentration of the target substance 110 in the reagent solution L210 is adjusted so that one molecule or less of the target substance enters the wells 241 per well.
  • a sealing liquid L220 is introduced into the fluidic device 500.
  • the specific gravity of the sealing liquid L220 is greater than that of the reagent liquid L210. Therefore, the sealing liquid L220 sinks below the reagent liquid L210 that is not contained in the wells 241 among the reagent liquids L210 and contacts the well array 240 . Then, the sealing liquid L220 individually seals the plurality of wells 241 containing the reagent liquid L210 containing the target substance to form independent reaction spaces (that is, minute compartments 242).
  • Microcompartment 242R is the well in which signal was detected and microcompartment 242 is the well in which no signal was detected.
  • the present invention provides a target substance 110, a first specific binding substance 120 to the target substance 110 labeled with the first single-stranded nucleic acid fragment 121, and a second single-stranded nucleic acid fragment. a second specific binding substance 130 to the target substance 110 labeled with 131, wherein at least a portion of the first single-stranded nucleic acid fragment 121 and at least a portion of the second single-stranded nucleic acid fragment 131 are hybridized; Composite 100 is provided that has been lysed to form double-stranded nucleic acid 140 .
  • a flap probe may further hybridize to the first single-stranded nucleic acid fragment 121 or the second single-stranded nucleic acid fragment 131.
  • the target substance can be detected without performing a washing step.
  • the present invention provides a kit for detecting a target substance 110, comprising a well array 240 having a plurality of wells 241, a first single-stranded nucleic acid fragment 121 labeled with a first and a second specific binding substance 130 to the target substance 110 labeled with a second single-stranded nucleic acid fragment 131.
  • the method for detecting the target substance described above can be suitably performed. Therefore, in the kit of the present embodiment, the well 241 contains the target substance 110, the first specific binding substance 120 for the target substance 110 labeled with the first single-stranded nucleic acid fragment 121, and the second one. A second specific binding substance 130 for a target substance 110 labeled with a single-stranded nucleic acid fragment 131 is introduced, resulting in a target substance 110, a first specific binding substance 120 and a second specific binding substance 130.
  • a step of forming a complex 100 containing and detecting the formation of the double-stranded nucleic acid 140, wherein the detection of the formation of the double-stranded nucleic acid 140 is for use in a method of indicating the presence of the target substance 110. can.
  • the well array may be arranged inside the fluidic device described above.
  • the target substance, the first single-stranded nucleic acid fragment, the first specific binding substance, the second single-stranded nucleic acid fragment, and the second specific binding substance are those described above. is similar to
  • the kit of this embodiment may further include a sealing liquid L220 that seals the opening of the well 241.
  • the sealing liquid L220 is the same as described above.
  • the kit of this embodiment detects a double-stranded nucleic acid 140 formed by hybridizing at least a portion of the first single-stranded nucleic acid fragment 121 and at least a portion of the second single-stranded nucleic acid fragment 131. Reagents may also be included.
  • reagents examples include the reagents for the ICA reaction described above, specifically flap probes, flap endonucleases (FEN), fluorescent substrates, and the like.
  • DNA1 (5'-TTTGTCACTGTTCCTCCTTTTGTTTTCCTTTCTGTGAGCAATTTCACCCAA-3', SEQ ID NO: 1) and DNA2 (5'-GCATGGTTCCAATTTGGGTGAT-3', SEQ ID NO: 2), which are oligonucleotides with an amino group modification at the 5' end, were bound respectively.
  • the length of the double-stranded nucleic acid formed by hybridization of DNA1 and DNA2 is 9 bases.
  • a target substance PSA antigen
  • the two types of oligonucleotide-modified antibodies described above, and a blocking buffer were mixed to prepare a mixed solution.
  • the volume of each mixture was 10 ⁇ L each.
  • the target substances were prepared to have final concentrations of 0 nM, 0.294 nM, 2.94 nM, 14.7 nM and 29.4 nM, respectively.
  • two types of oligonucleotide-modified antibodies were prepared to a final concentration of 8.56 nM, respectively.
  • As a blocking buffer Tris-buffered saline (TBS) containing 1% bovine serum albumin (BSA) was used. Subsequently, each mixture was allowed to react at 37° C. for 30 minutes.
  • ICA reaction reagent 1 An ICA reaction reagent for detection was prepared in order to carry out an ICA reaction using the oligonucleotide modified to the above antibody.
  • the ICA reaction reagents in this experimental example are 0.25 ⁇ M allele probe (5′-CGCGCCGAGGAATTGCTCACAGAAAGGA-3′) (Fasmac, SEQ ID NO: 3), 2 ⁇ M FRET cassette 1 (fluorescent substrate, Alexa488-BHQ: X-TTCT-Y -AGCCGGTTTTCCGGCTGAGACCTCGGCGCG, X: Alexa488 + AminoC6, Y: Black hole quencher (BHQ) 1-dT) (Nippon Bio Service Co., SEQ ID NO: 4), 0.81 ⁇ M flap endonuclease (FEN)-1, 50 mM Tris-HCl (pH 8. 5), 20 mM MgCl2 , and 0.0
  • FIG. 9 shows the results of immunoICA reaction using 0 nM, 0.294 nM, 2.94 nM, 14.7 nM and 29.4 nM of PSA antigen as target substances.
  • the vertical axis indicates the fluorescence intensity (relative value) of Alexa488, and the horizontal axis indicates the time (seconds) after the start of the ICA reaction.
  • the fluorescence intensity when the antigen concentration is 0 nM is N (noise), the fluorescence intensity at each other concentration is S (signal), and the signal-to-noise ratio (S/N ratio ) is a graph showing the calculation results.
  • the vertical axis indicates the S/N ratio
  • the horizontal axis indicates the time (seconds) after initiation of the ICA reaction.
  • the S/N ratio at an antigen concentration of 14.7 nM was 10.0 at 1080 seconds from the start of the reaction.
  • the S/N ratio at an antigen concentration of 29.4 nM was 5.6 at 1080 seconds from the start of the reaction.
  • the S/N ratio at an antigen concentration of 2.94 nM was 4.6 at 1380 seconds from the start of the reaction.
  • FIG. 11 is a graph showing the relationship between the antigen concentration (nM) and the S/N ratio 18 minutes after the start of the reaction.
  • the vertical axis indicates the S/N ratio
  • the horizontal axis indicates the antigen concentration (nM).
  • the highest S/N ratio was about 10 when the antigen concentration was 14.7 nM.
  • a fluorescence image of each well in the fluidic device was taken with a fluorescence microscope BZ-710 (KEYENCE) using a 10x objective lens.
  • the exposure time was 2 seconds using a GFP fluorescence filter.
  • Bright field images were also taken with a microscope BZ-710 using a 10x objective after sealing the wells.
  • the upper left of FIG. 12 is a bright field image at an antigen concentration of 0 pM.
  • the upper right of FIG. 12 is a fluorescence image at an antigen concentration of 0 pM.
  • the lower left of FIG. 12 is a bright field image at an antigen concentration of 14.7 nM.
  • the bottom right of FIG. 12 is a fluorescence image at an antigen concentration of 14.7 nM.
  • FIG. 13 is a graph showing the number of wells in which fluorescence was observed and the brightness when each concentration of target substance was detected.
  • the vertical axis indicates the number of wells in which fluorescence was observed
  • the horizontal axis indicates the luminance (relative value).
  • FIG. 14 is a graph showing the average value ⁇ standard deviation of luminance when target substances of each concentration are detected.
  • the vertical axis indicates luminance (relative value), and the horizontal axis indicates antigen concentration.
  • FIG. 15 is a graph showing the number of wells exhibiting brightness equal to or higher than the brightness threshold, which was temporarily set to 6000 in this experimental example, when a target substance of each concentration was detected. As a result, it was found that at an antigen concentration of 2.94 nM or more, approximately 3.7 times as many wells as those without antigen showed luminance above the threshold.
  • the length of the double-stranded nucleic acid formed by hybridizing DNA2 and DNA3 is 3 bases.
  • the length of the double-stranded nucleic acid formed by hybridizing DNA2 and DNA4 is 12 bases.
  • the length of the double-stranded nucleic acid formed by hybridization of DNA2 and DNA5 is 15 bases.
  • a mixture was prepared by mixing a target substance (PSA antigen), an oligonucleotide-modified antibody bound to DNA1 and DNA2, and a blocking buffer. The volume of each mixture was 10 ⁇ L each.
  • the target substance was prepared to have a final concentration of 0 nM or 2.94 nM.
  • two types of oligonucleotide-modified antibodies were prepared to a final concentration of 8.56 nM, respectively.
  • As a blocking buffer TBS containing 1% BSA was used. Subsequently, each mixture was allowed to react at 37° C. for 30 minutes.
  • modified oligonucleotide antibodies instead of the above combinations, a combination of oligonucleotide-modified antibodies bound to DNA2 and DNA3, respectively, a combination of oligonucleotide-modified antibodies bound to DNA2 and DNA4, respectively, and DNA2 and DNA5.
  • Mixtures were prepared in the same manner except that a combination of conjugated oligonucleotide-modified antibodies was used, and reactions were carried out under the same conditions.
  • ICA reaction reagent 2 An ICA reaction reagent for detection was prepared in order to carry out an ICA reaction using the oligonucleotide modified to the above antibody.
  • the ICA reaction reagents in this experimental example are 0.25 ⁇ M allele probe (5′-CGCGCCGAGGAATTGCTCACAGAAAGGA-3′) (Fasmac, SEQ ID NO: 3), 4 ⁇ M FRET cassette 1 (fluorescent substrate, Alexa488-BHQ: X-TTCT-Y -AGCCGGTTTTCCGGCTGAGACCTCGGCGCG, X: Alexa488 + AminoC6, Y: Black hole quencher (BHQ) 1-dT) (Nippon Bio Service Co., SEQ ID NO: 4), 0.81 ⁇ M flap endonuclease (FEN)-1, 50 mM Tris-HCl (pH 8. 5), 20 mM MgCl2 , and 0.05% Tween20.
  • FIG. 17 shows the results of immunoICA reaction using oligonucleotide-modified antibodies to which DNA2 and DNA3 were respectively bound.
  • FIG. 18 shows the results of immunoICA reaction using oligonucleotide-modified antibodies to which DNA2 and DNA4 were respectively bound.
  • FIG. 19 shows the results of immuno-ICA reaction using oligonucleotide-modified antibodies to which DNA2 and DNA5 were conjugated, respectively.
  • a mixed solution was prepared by mixing the target substance (PSA antigen), the oligonucleotide-modified antibody bound to DNA2 and DNA5, and the blocking buffer.
  • the volume of the mixture was 10 ⁇ L.
  • the target substance in the mixture was prepared to have a final concentration of 0 nM or 2.94 nM.
  • two types of oligonucleotide-modified antibodies were prepared to a final concentration of 8.56 nM, respectively. 10 ⁇ L of this mixture was mixed with 10 ⁇ L of the ICA reaction reagent prepared by the method described in ⁇ Preparation of ICA reaction reagent 2>>.
  • the final concentration of the target substance in the resulting mixture was 0 nM or 1.47 nM, and the final concentrations of the two types of oligonucleotide-modified antibodies were 4.28 nM, respectively.
  • This mixture was held at 37°C for 30 minutes for antigen-antibody reaction, then held at 66°C for 1 hour for immunoICA reaction, and Alexa488 fluorescence was detected using Rotor-Gene Q (QIAGEN). (Hereinafter referred to as reaction type II).
  • Fig. 20 shows the results of reaction type I and reaction type II. Even when compared with the method of mixing the ICA reaction solution after the antigen-antibody reaction as in Reaction Type I, there was no significant difference in immuno-ICA reaction intensity and background reaction intensity in Reaction Type II. From these results, it was found that the ICA reaction reagent does not attenuate the antigen-antibody reaction during the antigen-antibody reaction, and that the antigen-antibody reaction reagent does not attenuate the ICA reaction during the ICA reaction.
  • the antigen-antibody reaction and the immuno-ICA reaction can be performed successively, which simplifies the process, but it cannot be predicted that the immuno-ICA reaction can be detected with high sensitivity. It is an effect.

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